The term “template-directed synthesis” includes the formation of a new substance by chemical modification of a substrate, or by the coupling of two or more molecules in the presence of a template that serves as a pattern for new structure formation. The most well known example of this process is gene transcription. A particular example of template-directed synthesis is template polymerization, where the formation of a polymeric receptor (replica) proceeds in the presence of another polymer or small molecular weight organic substance (the template). Prior to the initiation of polymerization, and during polymerization, the monomers spatially distribute themselves (self-assembly process) around the template molecules in accordance with the size, polarity and functionality of the template. The monomers are polymerised either into linear chains or rigid three-dimensional networks. A specific example of template polymerization is molecular imprinting, based on the polymerization of vinyl or acrylic monomers in the presence of template (see ref. 1, 2). The traditional approach involves the production of highly cross-linked imprinted polymers, which are insoluble in aqueous and organic solvents. Because of their inherent insolubility, the possibility to use molecularly imprinted polymers (MIPs) in pharmacology and medicine is restricted.
Recently, several attempts have been made to develop protocols for the preparation of imprinted polymers with relatively low-molecular weights, which could exist in soluble or at least colloidal forms. This format will allow polymers to be used as biologically active molecules (drugs, effectors, modulators, inhibitors) in pharmacology and medicine, as “plastic antibodies”, replacing biological molecules in sensors and in affinity separation and as catalysts with enzyme-like properties.
In one such example, MIP molecules were synthesized by polycondensation of amino acids and nucleotides around a biological receptor, enzyme, nucleic acid, cell, virus, micro organism, tissue sample or drug (see U.S. Pat. No. 6,852,818). In another example, different methods were used to produce oligomeric and polymeric MIPs (see U.S. Pat. No. 6,127,154). Most of the examples in the prior art describe the preparation of high-molecular weight cross-linked polymers, which require hydrolysis in order to deliver soluble or colloidal particles stable in solution. In one such example, (see U.S. Pat. No. 6,127,154) researchers used specially designed compounds containing photoactive perfluorophenylazido groups capable of coupling upon illumination. In this case, affinity ligands could be synthesized as soluble particles. In all of these cases, the synthesized compounds were composed of a number of fractions with poorly controlled size and properties. Other approaches for the synthesis of molecules with biological activity are described in PCT Patent Publication WO 96/40822 and in U.S. Pat. No. 5,630,978, where chemical compounds were prepared in the presence of a template-imprinted polymer, which in turn was prepared in the presence of another template, normally a drug such as heparin. The resulting replica is a ligand molecule, which has no affinity to the template and rather resembles the structure of the original drug molecule itself.
One of the ways to produce nanoparticles is through the use of controlled condensation or additive radical “living” polymerization. The living free-radical polymerization techniques, such as iniferter polymerization, nitroxide-mediated radical polymerization, atom-transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization, open new routes for the synthesis of polymers with relatively controlled low-molecular weights (see ref. 3-9). Controlled/living polymerization techniques are based on a delicate balance between dormant and active species that effectively reduces the concentration of propagating entities in the system and minimizes the extent of termination. Living polymerization could be free of side reactions such as termination and chain transfer and thus can generate polymers with well-defined molecular weight distributions and structures. The same approach can be applied to the formation of copolymers, thus making it possible to produce block copolymers by free radical polymerization by proper sequencing of the monomer additions. Living polymerization has been used previously in the production of bulk grafted MIPs (see ref. 10, 11). Soluble polymers were also produced by living polymerization and used later in MIP production (see ref. 12). Recently, controlled living polymerization was used for the preparation of MIP nanoparticles (13).
One of the complications in MIP synthesis is the frequent need to use templates, which are expensive and/or difficult to obtain, such as proteins, some toxins etc. that are difficult to recover after polymerization and limit the amount of MIP that can be obtained. Ideally the template should be capable of being recycled to overcome these limitations. The optimal way to achieve this is by using the template in an immobilized form. Immobilized template has been used previously (see U.S. Pat. No. 7,393,909). In that case, the template was immobilized onto a silica surface and polymer was formed in pores around it. By dissolving the silica support and removal of the template, MIPs of various morphologies were obtained. In all of the examples disclosed in U.S. Pat. No. 7,393,909, the surface bearing the immobilized template is lost during the dissolution process and cannot be recycled. In other examples, immobilized templates were used for the production of imprinted surfaces (see U.S. Pat. Nos. 6,127,154; 6,458,599; and 7,288,415). Potentially the template-bearing surfaces disclosed in these reports can be regenerated and used several more times. These approaches can be used for the production of sensors or arrays, but would be difficult to adapt for the production of nanoparticles or small soluble molecules.
Yet another major problem associated with MIPs is the heterogeneity of the binding sites produced, which is generally responsible for high levels of non-specific binding. This problem has been rectified by affinity separation of separately produced MIP nanoparticles on a column with immobilized template (13). It is clear that in order for affinity separation to be possible, MIPs should be in a suitable form, preferably in the form of nanoparticles.
The current invention addresses all of these problems relating to the development of high performance cross-linked MIP nanoparticles by proposing a combination of two techniques: (i) performing controlled polymerization, optionally controlled radical polymerization, in the presence of a surface or surfaces bearing immobilized template to form imprinted nanoparticles and (ii) retaining the nanoparticles by affinity interaction with immobilized template for selection and purification purposes.
Background material can be found in the following references:    1. Wulff, G. Makromol. Chem. Macromol. Symp., 1993, 70/71, 285.    2. Vlatakis, G.; et al. Nature, 1993, 361, 645.    3. Moad, G.; Rizzardo E.; Solomon, D. H. Macromolecules 1982, 15, 909;    4. Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921.    5. Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. Rev. 2001, 101, 3689.    6. Hawker, C. J.; Bosman, A. W.; Harth, E. Chem. Rev. 2001, 101, 3661.    7. Fischer, H. Chem. Rev. 2001, 101, 3581.    8. Otsu, T.; Matsumoto, A. Adv. Polym. Sci. 1998, 136, 75-137.    9. Moad, G.; et al. Polym. Int. 2000, 49, 993-1001.    10. Ruckert, B.; Hall, A. J.; Sellergren B. J. Mater. Sci. 2002, 12, 2275.    11. Hattori, K.; et al. J. Membr. Sci. 2004, 233, 169.    12. Li, Z.; Day, M.; Ding, J. F.; Faid, K. Macromolecules. 2005, 38, 2620.    13. Guerreiro A. R., Chianella I., Piletska E., Whitcombe M. J., Piletsky S. A. (2009). Biosens. Bioelectron., 24, 2740-2743.    14. Jagur-Grodzinski, J. Reactive & Functional Polymers. 2001, 1, 1.    15. Shim, S. E. et al. Macromolecules. 2003, 36, 7994-8000.    16. Yu, Q.; Zeng, F.; Zhu S. Macromolecules. 2005, 34, 1612.    17. U.S. Pat. No. 7,019,072—Method of preparing latex for coating paper, 2006.
Additional patent references include:    1. U.S. Pat. No. 6,852,818 issued 8 Feb. 2005 entitled MOLECULARLY IMPRINTED POLYMERS PRODUCED BY TEMPLATE POLYMERIZATION.    2. U.S. Pat. No. 6,127,154 issued 3 Oct. 2000 entitled METHODS FOR DIRECT SYNTHESIS OF COMPOUNDS HAVING COMPLEMENTARY STRUCTURE TO A DESIRED MOLECULAR ENTITY AND USE THEREOF.    3. PCT Patent Publication WO96/40822 entitled PREPARATION OF BIOLOGICALLY ACTIVE MOLECULES BY MOLECULAR IMPRINTING.    4. U.S. Pat. No. 5,630,978 issued 20 May 1997 entitled PREPARATION OF BIOLOGICALLY ACTIVE MOLECULES BY MOLECULAR IMPRINTING.    5. U.S. Pat. No. 7,393,909 issued 1 Jul. 2008 entitled POROUS, MOLECULARLY IMPRINTED POLYMER AND A PROCESS FOR THE PREPARATION THEREOF.    6. U.S. Pat. No. 6,458,599 issued 1 Oct. 2002 entitled COMPOSITIONS AND METHODS FOR CAPTURING ISOLATING DETECTING ANALYZING AND QUANTIFYING MACROMOLECULES.    7. U.S. Pat. No. 7,288,415 issued 30 Oct. 2007 entitled COMPOSITIONS AND METHODS FOR CAPTURING ISOLATING DETECTING ANALYZING AND QUANTIFYING MACROMOLECULES.